RNA degradation - (Apr/18/2007 )
hi all, firstly thanks for your time. my problem is extraction of RNA from nematode's. i have done this successfully about 30 times. for the past month however i have had terrible trouble with degradation. i get no 28S or 18S bands when i run on my gel only degradation and a high 260/280 ratio about 2.2. also i find it hard to resuspend my precipatated pellet in DEPC h20 (not always). i wipe area thoroughly with RnAse away, i use brand new filter tips, buy in |DEPC h20, new trizol, chloroform, isopropanol and ethanol. i wash pestle with rnase away then double autoclave. two other lab members have this exact problem also so i doubt its technique. has anyone any ideas?
thank you
Not my field so forgive what may be a silly question but how are the nematodes stored prior to RNA isolation? are they alive? How do you kill them? are they stored in some place together with your collegues so that maybe a freezer that is at the wrong temperature or something is the cause? You didn't change the ratio of nematode to tri-reagent (if you use too little tri-reagent can be problem, or if you are now processing more samples so it sits longer before the isolation? If you really used brand new all the reagents you listed that should be fine so I guess I am moving backward to find another possible cause...
thanks for the reply! the nematode are firstly grown on NGM agar plates seeded with OP50 which they feed on. i wash them off with sterile h20, centrifuge to pellet then wash *3 with sterile H20. i then transfer pellet worms( still alive) to a new NGM plate with no op50 and leave overnight to crawl out. next morning cut out part of plate which had pellet on. rinse off now clean worms pellet add 1ml trizol snap frozen in liquid nitrogen. stored -80. we all have our own storage places for plates. -80 freezer checked regularly for temp. perhaps processing slightly more but i have reduced my pellet size and nothing happened.
could it be unseasonable heat we are having in ireland?
or perhaps a problem with our agarose?
Have you try to use any RNase inhibitor? I am pretty new for this RNA work as well.
Heat in ireland? Arent you doing in an air conditioned lab? Shouldnt be.
I am in a tropical country with heat all the year. I was able to extract RNA out from E.coli. But 2 weeks after that, everything is gone. I guess there is presence of RNase inhibitor.
You have a high ratio indicating presence of RNA. It is really weird. Initially I would thought perhaps your cells are not lysed properly. I know DTT or mercapto is able to inhibit RNAse activity by destroying the sulphide bond. But I am not sure which stage you should add while extracting the RNA out. Hope this helps.
Heat in ireland? Arent you doing in an air conditioned lab? Shouldnt be.
I am in a tropical country with heat all the year. I was able to extract RNA out from E.coli. But 2 weeks after that, everything is gone. I guess there is presence of RNase inhibitor.
You have a high ratio indicating presence of RNA. It is really weird. Initially I would thought perhaps your cells are not lysed properly. I know DTT or mercapto is able to inhibit RNAse activity by destroying the sulphide bond. But I am not sure which stage you should add while extracting the RNA out. Hope this helps.
hi! i have use B mercaptoethanol but to no avail.. we are beginning to come to the conclusion that it is our agarose gel that is the culprit. technicans not keeping an eye on cleanliness when distilling the h20 hence TAE gel may have RNases in it. i check my gel early in the run and bands seen. when i leave it longer to let the marker migrate degradation occurs. Its very unusual as anyone ever heard of this?
have you ever had my problem of resuspending the pellet? i resuspend in DEPC and incubate 55C for 10mins.
Perhaps you are right about the water used in the deionised water. It is really peculiar!
I don't have much problem resuspending pellet. Do you have a very high amount of pellet?
RNA pellet will be resuspended easily in RNAse free water (you can buy it from some companies).
And I suggest you check your DEPC treated water.
I am not sure if your problem is similar to mine. I am working with viral RNA. I found that after my RNA extraction, when I stored the RNA in RNase free water in -80oC, the RNA was degraded. However, there was once my PI told me to store it in isopropanol and keep in -80oC. I finished up my ethanol precipitation just before I run RT-PCR. Guess what? It works fantastic!!
hi all! thanks for the feedback! the results are in! it was the agarose gel made up with supposedly clean H20 that was degrading the RNA. so all the RNA that was thrown out, as degraded, was more than likely perfect! so word to the wise, keep an eye on your prep labs supposedly clean h20!
THANKS!
PS i always use DNase and RNAse free DEPC h2o for resuspension ,bought in from reputable companies. So that cannot be my problem for pellet not resuspending. perhaps it is to big a pellet though... no more ideas?